Genetics - Quantitation of Bacteriophage λ

Jeff Lawrence (G20)

1. Grow E. coli C600 overnight at 37° in 5 mL of liquid LB+ 0.1% Maltose. The maltose induces the lamB receptor gene. LB is:
	10 g 	Tryptone
	5 g 	Yeast Extract
	5 g 	NaCl
	1 L 	Water
2. Serially dilute λ bacteriophage in T2 buffer. First, make a 10-2 dilution by adding 10 µ λ bacteriophage stock to 1 mL T2 buffer.

3. Dilute serially by taking 0.1 mL of the previous dilution and placing it in 0.9 mL T2 buffer. Repeat until a 10-8 dilution is made. Final dilution depends upon the titer of the phage stock.

4. Mix 100 µL cells and 100 µL bacteriophage dilution. Incubate at 37° for 30 min to adsorb phage to cells.

5. Mix with 2.5 mL Top Agar. Make Top Agar as follows:
	1X	LB top agar	10 g	Tryptone
	5 mM	CaCl2		5 g	Yeast Extract
	10 mM	MgSO4		5 g	NaCl
				7 g	Agar
				5 mL	CaCl2
				10 mL	MgSO4
				985 mL	H2O

	a) Siphon solution into 100 mL autoclaved graduated cylinder 
	and dispense into 10 bottles while stirring.

	b) Autoclave for 20 min.
6. Vortex tube and quickly plate on λ plate warmed to room temperature. Make plates as:
	10 g	Tryptone
	5 g	NaCl
	15 g	Agar
	1 L	H2O
7. Incubate at 37° overnight.

9. Count plaques. Multiply the number of plaques by 10(1+Dilution) to calculate the titer of the original bacteriophage stock. For example:
 	200 plaques on the 10-7 dilution 
	yields (2 x 102 pfu/100 mL)*(10)*(107) = 2 x 1010 pfu/mL

Last Update: Thursday June 19 2014
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